Penn Medicine

Yale Goldman, M.D., Ph.D.

Fluorescence polarization transients from skeletal muscle fibers labeled with rhodamine isomers at SH-1 of the myosin heavy chain.

Berger CL, Craik JS, Trentham DR, Corrie JET, Goldman YE

Biophys. J. 71:3330-3343, 1996

Fluorescence polarization was used to examine orientational changes of rhodamine probes following photolysis of caged nucleotides and rapid length single, skinned muscle fibers from rabbit psoas muscle. Fibers were extensively and predominantly labeled at SH-1 (cys 707) of the myosin heavy chain with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine (IATR). Results from spectroscopic experiments utilizing the two probe isomers were quite similar. Following photolysis of either caged ATP or caged ADP, probes promptly reoriented toward the muscle fiber axis and became more disordered. The fluorescence polarization transients elicited by the photolysis of caged ATP in the presence of Ca2+ were slightly faster than the estimated rate of cross-bridge detachment and greatly preceded active force generation. Photolysis of caged ADP caused only a small, rapid decrease in force, but elicited changes in the fluorescence polarization signals with time course and amplitude similar to those following photolysis of caged ATP. Fluorescence polarization signals were virtually unchanged by rapid length steps in both rigor and active muscle fibers. These results suggest that structural changes at SH-1 monitored by 5- or 6-ATR are not associated directly with the force generating event of muscle contraction, but may be involved in the communication pathway between the nucleotide and actin-binding sites of myosin.

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Perelman School of Medicine University of Pennsylvania
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